Project Summary. Cysteine dioxygenase (CDO) is an Fe-containing tumor suppressor enzyme found to play a role in combatting a wide variety of human cancers, and nitrile hydratase (NHase) is an Fe-enzyme involved in the biosynthesis of antibiotics. Dioxygen (O2)-promoted Fe-S(R)-OH formation plays a key role in the mechanisms of both of these enzymes. The goal of the work proposed herein will be to elucidate the mechanisms of CDO and NHase, as well as the mechanism of posttranslational NHase sulfur oxidation. In addition we will determine why Isopenicillin N-synthase (IPNS), ovothiol synthase (OvoA), and CDO follow different reaction pathways. Like CDO, IPNS and OvoA are Fe enzymes that bind O2 cis to a thiolate, however the latter promote C?S bond formation, while CDO promotes S-O bond formation. The proposed project will involve the synthesis and spectroscopic characterization of small-molecule intermediate-analogues, and the T-dependent kinetics of oxo atom donor binding and activation, thiolate oxidation, and nitrile hydrolysis. Metastable cis-thiolate ligated Fe- O2, Fe-O2?, Fe-OOR, and Fe-OIAr intermediates recently prepared in our lab will be spectroscopically characterized, and we will determine their structures using calibrated DFT/TD-DFT or X-ray crystallography. Nitric oxide (NO) will be used to probe the O2 binding site of reduced cis-thiolate ligated Fe(II) complexes, and determine how the spin-state, and extent of N-O, and thus O-O, bond activation influence reaction pathways (HAT vs OAT to sulfur). Proton-induced addition of a 2nd oxo atom to our structurally characterized iron sulfenate (Fe-S(R)-O?) complex will be examined, and T-dependent kinetics will be used to determine the probable mechanism of ArIO-induced oxo atom addition to the cis thiolate, (RS)2Fe!RSFe-S(R)-O?. The kinetics and thermodynamics of oxo atom donor binding will be examined for a variety of para-substituted pX- ArIO (X= H, CF3, F, Me, Pr, cyclohexyl). Thermodynamic parameters (?H, ?S) for ArIO binding will be obtained from a van't Hoff plot, activation parameters (?H?, ?S?) will be obtained from an Eyring plot, and a Hammett plot will be generated. We will examine the KO2 and O2 reactivity of a more reactive Fe complex containing a modified (Et,Pr)-ligand, and Fe complexes that incorporate sterically encumbering di- isopropylphenyl (dipp)-groups, both of which should stabilize intermediates thereby facilitating spectroscopic characterization and crystallization. In addition, we will test our working hypothesis regarding the role of a highly conserved, catalytically essential 56Arg-NH3+ that is within H-bonding distance of the NHase RS-O? oxygen, and test the proposed mechanism for nitrile hydrolysis, which involves intramolecular attack by the RS- OH at a coordinated nitrile carbon. Our small molecules should facilitate the trapping, and spectroscopic characterization of a cyclic RS-O-C(R')=NH NHase intermediate analogue.